Spinal Afferents Research Articles

Intrathecal Fentanyl Abolishes the Exaggerated Pressor Response to Cycling Exercise in Never‐Treated Hypertensive Men

Animal and human studies report that the exercise pressor reflex-mediated increase in blood pressure (BP) is exaggerated in hypertensive subjects. Here we investigated the influence of spinal opioid receptor-sensitive muscle afferents on the BP response to cycling in patients with hypertension. Male subjects were divided into 3 groups: Never-treated hypertensive (UT-Hyper; n=5; 48±5 yrs; 149±6/96±4 mmHg), treated hypertensive (T-Hyper; n=3; 42±6 yrs; 141±8/84±5 mmHg), and normotensive (Norm; n=6; 47±4 yrs; 124±5/78±3 mmHg). Beat-to-beat intra-arterial BP and heart rate (HR) were monitored during leg cycling (40 W) for 120 s, before (CTRL) and after administration of intrathecal fentanyl (FENT; 50 μg, L2-L3 or L3-L4 space), attenuating the central projection of leg muscle afferent signal. We also assessed the BP response to handgrip exercise to rule out cephalic migration of FENT. UT-Hyper patients had an augmented BP response (+11±2 mmHg; P<0.01) during leg cycling compared with both T-Hyper (0±2 mmHg) and Norm groups (+4±2 mmHg). Following FENT, BP response of the UT-Hyper group was diminished (+6±2 mmHg; P<0.05) to a similar level to that of the Norm group. FENT neither affected the BP response of the Norm group (P=0.11), nor the HR response to cycling or the responses to handgrip of any group. These findings indicate that the exercise pressor reflex has an important role in mediating the exaggerated BP to exercise in never-treated hypertensive men. Grant Funding Source: Supported by the Brazilian Science Without Borders Program

Reduced Ability to Maintain Cardiac Output and Arterial Pressure During Coronary Artery Occlusion in Conscious Paraplegic Mice

Cardiovascular morbidity and mortality are high in individuals with spinal cord injuries (SCI) due to a relatively sedentary life style and higher prevalence of other cardiovascular disease risk factors including obesity and diabetes. Furthermore, the risk for cardiovascular disease is exacerbated by abnormal and unstable autonomic control of the heart and vasculature. In this context, the response to myocardial ischemia is complex and involves an excitatory sympathetic reflex that supports arterial pressure during ventricular injury and reductions in cardiac output. Specifically, cardiac spinal (sympathetic) afferents are excited by ischemic metabolites and elicit an excitatory sympathetic reflex which plays a major role in maintaining perfusion pressure. Individuals and animals with SCI at thoracic level 5 (T5X) maintain the cardio-cardiac sympathetic reflex but fail to excite sympathetic pre-ganglionic neurons below the level of the injury. Accordingly, the tolerance to coronary artery occlusion (CAO) was expected to be markedly reduced in T5X mice. As expected, T5X mice had a reduced ability to maintain cardiac output and arterial pressure during CAO. External lower body compression (20mmHg) markedly improved the tolerance to CAO by supporting arterial pressure and cardiac output. Results suggest that loss of sympathetic control below thoracic level 5 prevented the support of arterial pressure and cardiac output while external lower body compression markedly improved the tolerance to CAO in paraplegic mice. The results have the potential to impact the lives of individuals living with SCI by harnessing endogenous homeostatic control systems with a simple and easily administered therapeutic intervention.

Tu1878 Identification of Different Types of Spinal Afferent Nerve Endings That Encode Noxious and Innocuous Stimuli in the Stomach and Esophagus Using a Novel Anterograde Tracing Technique

Tu1878 Identification of Different Types of Spinal Afferent Nerve Endings That Encode Noxious and Innocuous Stimuli in the Stomach and Esophagus Using a Novel Anterograde Tracing Technique

Neural circuits underlying tongue movements for the prey-catching behavior in frog: distribution of primary afferent terminals on motoneurons supplying the tongue.

The hypoglossal motor nucleus is one of the efferent components of the neural network underlying the tongue prehension behavior of Ranid frogs. Although the appropriate pattern of the motor activity is determined by motor pattern generators, sensory inputs can modify the ongoing motor execution. Combination of fluorescent tracers were applied to investigate whether there are direct contacts between the afferent fibers of the trigeminal, facial, vestibular, glossopharyngeal-vagal, hypoglossal, second cervical spinal nerves and the hypoglossal motoneurons. Using confocal laser scanning microscope, we detected different number of close contacts from various sensory fibers, which were distributed unequally between the motoneurons innervating the protractor, retractor and inner muscles of the tongue. Based on the highest number of contacts and their closest location to the perikaryon, the glossopharyngeal-vagal nerves can exert the strongest effect on hypoglossal motoneurons and in agreement with earlier physiological results, they influence the protraction of the tongue. The second largest number of close appositions was provided by the hypoglossal and second cervical spinal afferents and they were located mostly on the proximal and middle parts of the dendrites of retractor motoneurons. Due to their small number and distal location, the trigeminal and vestibular terminals seem to have minor effects on direct activation of the hypoglossal motoneurons. We concluded that direct contacts between primary afferent terminals and hypoglossal motoneurons provide one of the possible morphological substrates of very quick feedback and feedforward modulation of the motor program during various stages of prey-catching behavior.

Identification of different types of spinal afferent nerve endings that encode noxious and innocuous stimuli in the large intestine using a novel anterograde tracing technique.

In mammals, sensory stimuli in visceral organs, including those that underlie pain perception, are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRG). One of the major challenges in visceral organs has been how to identify the different types of nerve endings of spinal afferents that transduce sensory stimuli into action potentials. The reason why spinal afferent nerve endings have been so challenging to identify is because no techniques have been available, until now, that can selectively label only spinal afferents, in high resolution. We have utilized an anterograde tracing technique, recently developed in our laboratory, which facilitates selective labeling of only spinal afferent axons and their nerve endings in visceral organs. Mice were anesthetized, lumbosacral DRGs surgically exposed, then injected with dextran-amine. Seven days post-surgery, the large intestine was removed. The characteristics of thirteen types of spinal afferent nerve endings were identified in detail. The greatest proportion of nerve endings was in submucosa (32%), circular muscle (25%) and myenteric ganglia (22%). Two morphologically distinct classes innervated myenteric ganglia. These were most commonly a novel class of intraganglionic varicose endings (IGVEs) and occasionally rectal intraganglionic laminar endings (rIGLEs). Three distinct classes of varicose nerve endings were found to innervate the submucosa and circular muscle, while one class innervated internodal strands, blood vessels, crypts of lieberkuhn, the mucosa and the longitudinal muscle. Distinct populations of sensory endings were CGRP-positive. We present the first complete characterization of the different types of spinal afferent nerve endings in a mammalian visceral organ. The findings reveal an unexpectedly complex array of different types of primary afferent endings that innervate specific layers of the large intestine. Some of the novel classes of nerve endings identified must underlie the transduction of noxious and/or innocuous stimuli from the large intestine.

Influence of common synaptic input to motor neurons on the neural drive to muscle in essential tremor.

Tremor in essential tremor (ET) is generated by pathological oscillations at 4-12 Hz, likely originating at cerebello-thalamo-cortical pathways. However, the way in which tremor is represented in the output of the spinal cord circuitries is largely unknown because of the difficulties in identifying the behavior of individual motor units from tremulous muscles. By using novel methods for the decomposition of multichannel surface EMG, we provide a systematic analysis of the discharge properties of motor units in nine ET patients, with concurrent recordings of EEG activity. This analysis allowed us to infer the contribution of common synaptic inputs to motor neurons in ET. Motor unit short-term synchronization was significantly greater in ET patients than in healthy subjects. Furthermore, the strong association between the degree of synchronization and the peak in coherence between motor unit spike trains at the tremor frequency indicated that the high synchronization levels were generated mainly by common synaptic inputs specifically at the tremor frequency. The coherence between EEG and motor unit spike trains demonstrated the presence of common cortical input to the motor neurons at the tremor frequency. Nonetheless, the strength of this input was uncorrelated to the net common synaptic input at the tremor frequency, suggesting a contribution of spinal afferents or secondary supraspinal pathways in projecting common input at the tremor frequency. These results provide the first systematic analysis of the neural drive to the muscle in ET and elucidate some of its characteristics that determine pathological tremulous muscle activity.

Spinal μ-opioid receptor-sensitive lower limb muscle afferents determine corticospinal responsiveness and promote central fatigue in upper limb muscle.

We investigated the influence of group III/IV lower limb muscle afferents on the development of supraspinal fatigue and the responsiveness of corticospinal projections to an arm muscle. Eight males performed constant-load leg cycling exercise (80% peak power output) for 30 s (non-fatiguing) and to exhaustion (∼9 min; fatiguing) both under control conditions and with lumbar intrathecal fentanyl impairing feedback from μ-opioid receptor-sensitive lower limb muscle afferents. Voluntary activation (VA) of elbow flexors was assessed via transcranial magnetic stimulation (TMS) during maximum voluntary contraction (MVC) and corticospinal responsiveness was monitored via TMS-evoked potentials (MEPs) during a 25% MVC. Accompanied by a significant 5 ± 1% reduction in VA from pre- to post-exercise, elbow flexor MVC progressively decreased during the fatiguing trial (P < 0.05). By contrast, with attenuated feedback from locomotor muscle afferents, MVC and VA remained unchanged during fatiguing exercise (P > 0.3). MEPs decreased by 36 ± 6% (P < 0.05) from the start of exercise to exhaustion under control conditions, but this reduction was prevented with fentanyl blockade. Furthermore, fentanyl blockade prevented the significant increase in elbow flexor MEP observed from rest to non-fatiguing exercise under control conditions and resulted in a 14% lower corticospinal responsiveness during this short bout (P < 0.05). Taken together, in the absence of locomotor muscle fatigue, group III/IV-mediated leg muscle afferents facilitate responsiveness of the motor pathway to upper limb flexor muscles. By contrast, in the presence of cycling-induced leg fatigue, group III/IV locomotor muscle afferents facilitate supraspinal fatigue in remote muscle not involved in the exercise and disfacilitate, or inhibit, the responsiveness of corticospinal projections to upper limb muscles.

Innervation of enteric mast cells by primary spinal afferents in guinea pig and human small intestine.

Mast cells express the substance P (SP) neurokinin 1 receptor and the calcitonin gene-related peptide (CGRP) receptor in guinea pig and human small intestine. Enzyme-linked immunoassay showed that activation of intramural afferents by antidromic electrical stimulation or by capsaicin released SP and CGRP from human and guinea pig intestinal segments. Electrical stimulation of the afferents evoked slow excitatory postsynaptic potentials (EPSPs) in the enteric nervous system. The slow EPSPs were mediated by tachykinin neurokinin 1 and CGRP receptors. Capsaicin evoked slow EPSP-like responses that were suppressed by antagonists for protease-activated receptor 2. Afferent stimulation evoked slow EPSP-like excitation that was suppressed by mast cell-stabilizing drugs. Histamine and mast cell protease II were released by 1) exposure to SP or CGRP, 2) capsaicin, 3) compound 48/80, 4) elevation of mast cell Ca²⁺ by ionophore A23187, and 5) antidromic electrical stimulation of afferents. The mast cell stabilizers cromolyn and doxantrazole suppressed release of protease II and histamine when evoked by SP, CGRP, capsaicin, A23187, electrical stimulation of afferents, or compound 48/80. Neural blockade by tetrodotoxin prevented mast cell protease II release in response to antidromic electrical stimulation of mesenteric afferents. The results support a hypothesis that afferent innervation of enteric mast cells releases histamine and mast cell protease II, both of which are known to act in a diffuse paracrine manner to influence the behavior of enteric nervous system neurons and to elevate the sensitivity of spinal afferent terminals.

Activation of Hindbrain Neurons Is Mediated by Portal-Mesenteric Vein Glucosensors During Slow-Onset Hypoglycemia

Hypoglycemic detection at the portal-mesenteric vein (PMV) appears mediated by spinal afferents and is critical for the counter-regulatory response (CRR) to slow-onset, but not rapid-onset, hypoglycemia. Since rapid-onset hypoglycemia induces Fos protein expression in discrete brain regions, we hypothesized that denervation of the PMV or lesioning spinal afferents would suppress Fos expression in the dorsal medulla during slow-onset hypoglycemia, revealing a central nervous system reliance on PMV glucosensors. Rats undergoing PMV deafferentation via capsaicin, celiac-superior mesenteric ganglionectomy (CSMG), or total subdiaphragmatic vagotomy (TSV) were exposed to hyperinsulinemic–hypoglycemic clamps where glycemia was lowered slowly over 60–75 min. In response to hypoglycemia, control animals demonstrated a robust CRR along with marked Fos expression in the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus. Fos expression was suppressed by 65–92% in capsaicin-treated animals, as was epinephrine (74%), norepinephrine (33%), and glucagon (47%). CSMG also suppressed Fos expression and CRR during slow-onset hypoglycemia, whereas TSV failed to impact either. In contrast, CSMG failed to impact upon Fos expression or the CRR during rapid-onset hypoglycemia. Peripheral glucosensory input from the PMV is therefore required for activation of hindbrain neurons and the full CRR during slow-onset hypoglycemia.

Identification of bladder and colon afferents in the nodose ganglia of male rats.

The sensory neurons innervating the urinary bladder and distal colon project to similar regions of the central nervous system and often are affected simultaneously by various diseases and disorders, including spinal cord injury. Anatomical and physiological commonalities between the two organs involve the participation of shared spinally derived pathways, allowing mechanisms of communication between the bladder and colon. Prior electrophysiological data from our laboratory suggest that the bladder also may receive sensory innervation from a nonspinal source through the vagus nerve, which innervates the distal colon as well. The present study therefore aimed to determine whether anatomical evidence exists for vagal innervation of the male rat urinary bladder and to assess whether those vagal afferents also innervate the colon. Additionally, the relative contribution to bladder and colon sensory innervation of spinal and vagal sources was determined. By using lipophilic tracers, neurons that innervated the bladder and colon in both the nodose ganglia (NG) and L6/S1 and L1/L2 dorsal root ganglia (DRG) were quantified. Some single vagal and spinal neurons provided dual innervation to both organs. The proportions of NG afferents labeled from the bladder did not differ from spinal afferents labeled from the bladder when considering the collective population of total neurons from either group. Our results demonstrate evidence for vagal innervation of the bladder and colon and suggest that dichotomizing vagal afferents may provide a neural mechanism for cross-talk between the organs.

Gephyrin clusters are absent from small diameter primary afferent terminals despite the presence of GABA(A) receptors.

Whereas both GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs) play a role in control of dorsal horn neuron excitability, their relative contribution to inhibition of small diameter primary afferent terminals remains controversial. To address this, we designed an approach for quantitative analyses of the distribution of GABA(A)R-subunits, GlyR α1-subunit and their anchoring protein, gephyrin, on terminals of rat spinal sensory afferents identified by Calcitonin-Gene-Related-Peptide (CGRP) for peptidergic terminals, and by Isolectin-B4 (IB4) for nonpeptidergic terminals. The approach was designed for light microscopy, which is compatible with the mild fixation conditions necessary for immunodetection of several of these antigens. An algorithm was designed to recognize structures with dimensions similar to those of the microscope resolution. To avoid detecting false colocalization, the latter was considered significant only if the degree of pixel overlap exceeded that expected from randomly overlapping pixels given a hypergeometric distribution. We found that both CGRP(+) and IB4(+) terminals were devoid of GlyR α1-subunit and gephyrin. The α1 GABA(A)R was also absent from these terminals. In contrast, the GABA(A)R α2/α3/α5 and β3 subunits were significantly expressed in both terminal types, as were other GABA(A)R-associated-proteins (α-Dystroglycan/Neuroligin-2/Collybistin-2). Ultrastructural immunocytochemistry confirmed the presence of GABA(A)R β3 subunits in small afferent terminals. Real-time quantitative PCR (qRT-PCR) confirmed the results of light microscopy immunochemical analysis. These results indicate that dorsal horn inhibitory synapses follow different rules of organization at presynaptic versus postsynaptic sites (nociceptive afferent terminals vs inhibitory synapses on dorsal horn neurons). The absence of gephyrin clusters from primary afferent terminals suggests a more diffuse mode of GABA(A)-mediated transmission at presynaptic than at postsynaptic sites.

Neurobiology of visceral pain

Visceral pain is diffusely localized, referred into other tissues, frequently not correlated with visceral traumata, preferentially accompanied by autonomic and somatomotor reflexes, and associated with strong negative affective feelings. It belongs together with the somatic pain sensations and non-painful body sensations to the interoception of the body. (1) Visceral pain is correlated with the excitation of spinal (thoracolumbar, sacral) visceral afferents and (with a few exceptions) not with the excitation of vagal afferents. Spinal visceral afferents are polymodal and activated by adequate mechanical and chemical stimuli. All groups of spinal visceral afferents can be sensitized (e.g., by inflammation). Silent mechanoinsensitive spinal visceral afferents are recruited by inflammation. (2) Spinal visceral afferent neurons project into the laminae I, II (outer part IIo) and V of the spinal dorsal horn over several segments, medio-lateral over the whole width of the dorsal horn and contralateral. Their activity is synaptically transmitted in laminae I, IIo and deeper laminae to viscero-somatic convergent neurons that receive additionally afferent synaptic (mostly nociceptive) input from the skin and from deep somatic tissues of the corresponding dermatomes, myotomes and sclerotomes. (3) The second-order neurons consist of excitatory and inhibitory interneurons (about 90 % of all dorsal horn neurons) and tract neurons activated monosynaptically in lamina I by visceral afferent neurons and di- or polysynaptically in deeper laminae. (4) The sensitization of viscero-somatic convergent neurons (central sensitization) is dependent on the sensitization of spinal visceral afferent neurons, local spinal excitatory and inhibitory interneurons and supraspinal endogenous control systems. The mechanisms of this central sensitization have been little explored. (5) Viscero-somatic tract neurons project through the contralateral ventrolateral tract and presumably other tracts to the lower and upper brain stem, the hypothalamus and via the thalamus to various cortical areas. (6) Visceral pain is presumably (together with other visceral sensations and nociceptive as well as non-nociceptive somatic body sensations) primarily represented in the posterior dorsal insular cortex (primary interoceptive cortex). This cortex receives in primates its spinal synaptic inputs mainly from lamina I tract neurons via the ventromedial posterior nucleus of the thalamus. (7) The transmission of activity from visceral afferents to second-order neurons in spinal cord is modulated in an excitatory and inhibitory way by endogenous anti- and pronociceptive control systems in the lower and upper brain stem. These control systems are under cortical control. (8) Visceral pain is referred to deep somatic tissues, to the skin and to other visceral organs. This referred pain consists of spontaneous pain and mechanical hyperalgesia. The mechanisms underlying referred pain and the accompanying tissue changes have been little explored.

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Urinary bladder distention evoked visceromotor responses as a model for bladder pain in mice.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Cortical Neuron Response Properties Are Related to Lesion Extent and Behavioral Recovery after Sensory Loss from Spinal Cord Injury in Monkeys

Lesions of the dorsal columns at a mid-cervical level render the hand representation of the contralateral primary somatosensory cortex (area 3b) unresponsive. Over weeks of recovery, most of this cortex becomes responsive to touch on the hand. Determining functional properties of neurons within the hand representation is critical to understanding the neural basis of this adaptive plasticity. Here, we recorded neural activity across the hand representation of area 3b with a 100-electrode array and compared results from owl monkeys and squirrel monkeys 5-10 weeks after lesions with controls. Even after extensive lesions, performance on reach-to-grasp tasks returned to prelesion levels, and hand touches activated territories mainly within expected cortical locations. However, some digit representations were abnormal, such that receptive fields of presumably reactivated neurons were larger and more often involved discontinuous parts of the hand compared with controls. Hand stimulation evoked similar neuronal firing rates in lesion and control monkeys. By assessing the same monkeys with multiple measures, we determined that properties of neurons in area 3b were highly correlated with both the lesion severity and the impairment of hand use. We propose that the reactivation of neurons with near-normal response properties and the recovery of near-normal somatotopy likely supported the recovery of hand use. Given the near-completeness of the more extensive dorsal column lesions we studied, we suggest that alternate spinal afferents, in addition to the few spared primary axon afferents in the dorsal columns, likely have a major role in the reactivation pattern and return of function.

Alteration of nerve growth factor in dorsal root ganglia at early time of acute myocardial infarction and the role of spinal nerve afferents

Nerve growth factor (NGF) plays important roles in transmission of nociception, neural innervation and survival in sympathetic and sensory neurons. Evidence indicates that NGF may sensitize the sensory and sympathetic reaction upon noxious stimulation. Understanding of the alterations of NGF in the sensory neurons during acute myocardial infarction and the underlying mechanism may promote clinical prognosis of the pathology. The aim of the study was to investigate the changes of NGF in the sensory neurons innervating the heart at early time after myocardial infarction and potential role of afferent nerve signals in modulation of NGF. The myocardial infarction was induced by ligation of the left anterior descending branch of coronary artery in anesthetized rats. The expressions of NGF and its coding mRNA in the sensory neurons in the dorsal root ganglia of upper thoracic segments (1–5), with and without prior blockade of the spinal nerves, were examined using immunohistochemical assay, enzyme-linked immunosorbent assay and real-time quantitative reverse transcription polymerase chain reaction assay. It was found that the immunoreactive material for NGF was significantly increased in the ganglia (P<0.05) at 60min of myocardial infarction, without change in NGF mRNA before and after the time. Blockade of the spinal nerves obviously inhibited the expression of NGF (P<0.05) and the coding mRNA (P<0.01). The results may indicate that the spinal nerve afferents are important in sustaining and up-regulating the expression of NGF in the sensory neurons innervating the heart in acute myocardial infarction.

A novel anterograde neuronal tracing technique to selectively label spinal afferent nerve endings that encode noxious and innocuous stimuli in visceral organs

One major weakness in our understanding of pain perception from visceral organs is the lack of knowledge of the location, morphology and neurochemistry of all the different types of spinal afferent nerve endings, which detect noxious and innocuous stimuli. This is because we lack techniques to selectively label only spinal afferents. Our aim was to develop an anterograde tracing technique that labels only spinal afferent nerve endings in visceral organs, without also labeling all other classes of extrinsic afferent and efferent nerves. Mice were anesthetized with isoflurane and dextran-biotin injected, via glass micropipettes (diameter 5 μm), into L6 and S1 dorsal root ganglia. Mice recovered for 7 days, were then euthanized and the colon removed. Anterograde labeling revealed multiple unique classes of afferent endings that terminated within distinct anatomical layers of the colon and rectum. We characterized a particular class of intramuscular ending in the circular muscle (CM) layer of the colon that consists of multiple varicose axons that project circumferentially. We demonstrate a technique for selective anterograde labeling of spinal afferent nerve endings in visceral organs. This approach facilitates selective visualization of the precise morphology and location of the different classes of spinal afferent endings, without visual interference caused by indiscriminant labeling of other classes of afferent and efferent nerve axons which also innervate internal organs. We have used this new technique to identify and describe the details of a particular class of intramuscular spinal afferent ending in the CM layer of mouse large intestine.

Localization of receptors for calcitonin-gene-related peptide to intraganglionic laminar endings of the mouse esophagus: peripheral interaction between vagal and spinal afferents?

The calcitonin-gene-related peptide (CGRP) receptor is a heterodimer of calcitonin-receptor-like receptor (CLR) and receptor-activity-modifying protein 1 (RAMP1). Despite the importance of CGRP in regulating gastrointestinal functions, nothing is known about the distribution and function of CLR/RAMP1 in the esophagus, where up to 90% of spinal afferent neurons contain CGRP. We detected CLR/RAMP1 in the mouse esophagus using immunofluorescence and confocal laser scanning microscopy and examined their relationship with neuronal elements of the myenteric plexus. Immunoreactivity for CLR and RAMP1 colocalized with VGLUT2-positive intraganglionic laminar endings (IGLEs), which were contacted by CGRP-positive varicose axons presumably of spinal afferent origin, typically at sites of CRL/RAMP1 immunoreactivity. This provides an anatomical basis for interaction between spinal afferent fibers and IGLEs. Immunoreactive CLR and RAMP1 also colocalized in myenteric neurons. Thus, CGRP-containing spinal afferents may interact with both vagal IGLEs and myenteric neurons in the mouse esophagus, possibly modulating motility reflexes and inflammatory hypersensitivity.

The development of fibrosis in a novel model of chronic pancreatitis is mediated by complement factor C5

The development of fibrosis in a novel model of chronic pancreatitis is mediated by complement factor C5

Mechanisms involved in abdominal nociception induced by either TRPV1 or TRPA1 stimulation of rat peritoneum

Abdominal pain is a frequent symptom of peritoneal cavity irritation, but little is known about the role of the receptors for irritant substances, transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), in this painful condition. Thus, we investigated the abdominal nociception caused by peritoneal stimulation with TRPV1 (capsaicin) and TRPA1 (allyl isothiocyanate, AITC) agonists and their mechanisms in rats. The intraperitoneal (i.p.) injection of either capsaicin or AITC (0.03–10mg/kg) induced short-term (up to 20min) and dose-dependent abdominal nociception, and also produced c-fos expression in spinal afferents of the dorsal horn. TRPV1 antagonism prevented (94±4% inhibition) nociception induced by capsaicin but not by AITC. In contrast, the TRPA1 antagonism almost abolished AITC-induced nociception (95±2% inhibition) without altering the capsaicin response. Moreover, nociception induced by either capsaicin or AITC was reduced by the desensitisation of TRPV1-positive sensory fibres with resiniferatoxin (73±18 and 76±15% inhibitions, respectively) and by the NK1 receptor antagonist aprepitant (56±5 and 53±8% inhibitions, respectively). Likewise, the i.p. injections of capsaicin or AITC increased the content of substance P in the peritoneal fluid. Nevertheless, neither the mast cell membrane stabiliser cromoglycate, nor the H1 antagonist promethazine, nor depletion of peritoneal macrophages affected abdominal nociception induced either by capsaicin or AITC. Accordingly, neither capsaicin nor AITC increased the histamine content in the peritoneal fluid or provoked peritoneal mast cell degranulation in vitro. Collectively, our findings suggest that TRPV1 and TRPA1 stimulation in the peritoneum produces abdominal nociception that is mediated by sensory fibres activation.